EP0260148A2 - Méthode d'expression recombinante, vecteur et cellules transformées - Google Patents

Méthode d'expression recombinante, vecteur et cellules transformées Download PDF

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EP0260148A2
EP0260148A2 EP87308060A EP87308060A EP0260148A2 EP 0260148 A2 EP0260148 A2 EP 0260148A2 EP 87308060 A EP87308060 A EP 87308060A EP 87308060 A EP87308060 A EP 87308060A EP 0260148 A2 EP0260148 A2 EP 0260148A2
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sequence
cells
factor viii
expression
vector
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EP0260148A3 (fr
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Cornelia Maxine Gorman
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Genentech Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/64—Relaxins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21069—Protein C activated (3.4.21.69)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/44—Vectors comprising a special translation-regulating system being a specific part of the splice mechanism, e.g. donor, acceptor

Definitions

  • This invention relates to the application of recombinant DNA technology to prepare vectors capable of expressing desired proteins such that continuous production of the protein can be achieved. Furthermore, the invention relates to the construction of an expression vector capable of generating stable cytoplasmic mRNA so as to give rise to continuous production of the desired protein. In another aspect the invention relates to an expression vector having a specific stabilizing sequence positioned 5 ⁇ to a DNA encoding a desired protein. The invention further relates to the transfection of eukaryotic cells with such vectors and choosing of a host cell such that continuous production of the desired protein by that cell line is established.
  • eukaryotic cells specifically mammalian cells were transformed with heterologous DNA coding for a selectable phenotype. Wigler, ⁇ M., et al ., Cell 11: 223-232 (1977). It has also been shown that eukaryotic cells can be transformed to yield transformants having heterologous DNA integrated into the chromosomal DNA of the eukaryotic cell nucleus.
  • RNA polymerase makes a primary transcript of the entire DNA, both exons and introns. This transcript was processed so that the introns were removed while at the same time the exons were all joined together in the correct order. The mechanism producing the foregoing result is referred to as "splicing.”
  • introns exist in virtually all mammalian and vertebrate genes and also in the genes of eukaroytic microorganisms. Introns are not limited to the coding region of a message. For example, one intron was found in the leader region of the plasminogen activator mRNA before the coding sequence in addition to multiple splice sites elsewhere in the gene. Fisher, R. et al ., J. Biol. Chem. 260 , 1122 (1985). There has been considerable speculation about why introns have evolved and become such a general feature of eukaryotic genes. Crick, F., Science 204 , 264, 1979; and, Sharp, P.A., Cell 23 , 643-646 (1981).
  • RNA splicing plays in gene expression
  • Hamer, D.H. and Leder, P., Cell 18 , 1299-1302 (1979) manipulated the location and/or presence of a splice site in SV40 recombinants transfected into monkey cells.
  • Hamer and Leder, supra used one splice site located within the gene encoding the desired protein or used two splice site sequences, one located 5 ⁇ to and the second within the gene encoding the desired protein. They found that RNA were produced transiently by all of the viruses that retain at least one functional splice junction. They concluded that splicing is a prerequisite for stable RNA formation. Confirming that result, Gruss, P. et al.
  • the Gruss paper utilized a multipartite leader having several splice site sequences.
  • the three papers discussed all utilize viral vectors with numerous splice sites at various locations. These viral vectors differ from the nonviral vectors of the instant invention in several respects.
  • First viral introns are located both 5 ⁇ and 3 ⁇ to the transcription unit as well as within the coding sequence itself.
  • the stabilizing sequence is located 5 ⁇ to the gene encoding the desired protein.
  • Viral vectors continue to replicate independent of the host DNA, do not integrate and are lytic. Finally, many viral vectors require early gene function for correct splicing to occur.
  • the tripartite leader region is spliced onto multiple coding regions to yield a final message.
  • the complexity of the splicing pattern is evident from the fact that 4 primary transcripts can be differentially spliced to yield 14 discrete messages.
  • the controls for selection of downstream splicing to the coding sequence is not well understood. However, selection of the appropriate polyadenylation site and transcription termination precede the final splicing event and may effect the selection of the 3 ⁇ splice site.
  • vectors containing the adenomajor late promoter were constructed using cDNA's from other proteins such as DHFR or t-PA with the identical 5 ⁇ and 3 ⁇ control regions as described in European Patent Publication No. 160,457.
  • an expression vector having: an SV40 enhancer; the adenovirus major late promoter and tripartite leader sequences; a hybrid intron consisting of a 5 ⁇ splice site from the first exon of the tripartite leader and a 3 ⁇ splice site from a mouse immunoglobulin gene; two cDNAs the first encoding a desired protein, colony stimulating factor, and the second DHFR; SV40 polyadenylation sequence; and, VA gene.
  • This polycistronic vector was found to work only transiently, supra at 810, required the presence of VA RNA to increase translatability, supra at 811, and required a second cDNA, that of DHFR, to increase mRNA stability. supra at 811. So while a restricted transient expression capability was seen with adenovirus major late vectors which included the entire tripartite leader for some proteins, certain proteins have additional requirements for successful continuous expression.
  • a vector was constructed containing a cytomegalovirus promoter and enhancer, a cDNA encoding factor VIII, and a 3 ⁇ terminating sequence, absent any intron or constructed splice site. Neither transient nor stable expression of factor VIII was observed in any of the cell types tested.
  • Another vector absent an intron or constructed splice site containing the SV40 early transcriptional sequences including the enhancer and promoter, cDNA encoding factor VIII and the SV40 polyadenylation site produced neither transient nor stable expression.
  • a vector containing the SV40 promoter and enhancer, the entire adenomajor late tripartite leader i.e. three introns with appropriate donor and acceptor sites, cDNA encoding factor VIII and the 3 ⁇ hepatitis surface antigen polyadenylation site produced transient expression of factor VIII only in COS cells but no other cell types.
  • Ig immunoglobulin
  • a vector was constructed containing an SV40 enhancer and promoter, the first donor site and intron of the adenomajor tripartite leader, the consensus sequence for the Ig variable region acceptor sequence, the cDNA encoding factor VIII and the 3 ⁇ polyadenylation site of the hepatitis surface antigen. This vector failed to provide transient or stable expression of express factor VIII.
  • Yet another vector was constructed comprising the SV40 enhancer and promoter, cDNA encoding factor VIII, an SV40 small t-­antigen intron 3 ⁇ to the cDNA, complete with donor and acceptor sequence and the SV40 early region polyadenylation site. This vector failed to produce either transient or stable expression of factor VIII in any cell type.
  • the present invention is directed to the construction and use of vectors having a specific stabilizing sequence positioned 5 ⁇ to the DNA encoding certain proteins that are difficult to express.
  • the expression vector of the instant invention when transfected into a selected host cell will transfrom that host cell to one that provides continuous production of a desired protein, e.g. factor VIII.
  • the invention is also directed to the choice of an appropriate cell line and transfection of that host cell to establish a cell line for continuous production of the desired protein.
  • the present invention is based on the discovery that continuous production of some proteins by use of a recombinant expression vector requires a particular arrangement of a stabilizing sequence, located 5 ⁇ to the DNA encoding the desired protein. Furthermore, the invention relates to a stable cytoplasmic mRNA resulting from use of a stabilizing sequence positioned 5 ⁇ to a DNA encoding a desired protein. In another aspect the invention is directed to the expression vectors constructed in accord with the foregoing which express the gene encoding the desired heterologous protein.
  • the invention relates to the choice of an appropriate host cell for transfection with the novel vector of the instant invention.
  • Yet another aspect of the instant invention is the transformation of a host cell to establish a stable cell line for production of the desired heterologous protein.
  • nucleotide sequence refers to a nucleic acid comprising a series of nucleotides in a 5 ⁇ to 3 ⁇ phosphate diester linkage which may be either an RNA or a DNA sequence. If a DNA, the nucleotide sequence may be either single or double stranded. Similarly, “DNA sequence” refers to both single and double stranded embodiments.
  • Desired heterologous protein refers to a protein which is desired to be expressed in a host cell, but which the host cell either normally does not produce itself or produces in small amounts, and which is not normally necessary for the cells continued existence.
  • a protein includes any molecule having the pre or mature amino acid sequence and amino acid or glycosylation variants (including alleles) capable of exhibiting a biological activity in common with said desired heterologous protein.
  • Splicing refers to the mechanism by which a single functional RNA molecule is produced by the removal of one or more internal stretches of RNA during the processing of the primary transcript. Splicing is believed to begin with the looping out of the intron so that the 5 ⁇ end of the intron (referred to as the donor) is juxtaposed to the 3 ⁇ end of the intron (referred to as the acceptor). A comparison of the base sequences at intron-exon junctions reveals consensus sequences, with the first two bases at the 5 ⁇ end of each intron being GT and the last two bases at the 3 ⁇ end being AG.
  • spliced mRNA refers herein to mRNA produced by either the removal of one or more internal stretches of RNA or by constructing a DNA which when transcribed produces a mRNA having the same properties as a mRNA which had been subject to splicing but from which no nucleotide sequence had in fact been removed.
  • Stabilizing sequence refers to a DNA sequence that gives rise to a spliced mRNA by coding either a splice donor-intron-­acceptor sequence or by coding a sequence comprising a full consensus sequence or a part thereof for the donor and acceptor sequence and the appropriate nucleotides at the donor/acceptor junction such that the resulting mRNA resembles functionally a mRNA which had been spliced.
  • the stabilizing sequence is placed in the leader sequence of the gene encoding the desired heterologous protein.
  • Leader sequence refers to that region of mRNA that is in the 5 ⁇ untranslated region between the CAP site and the AUG translation start signal.
  • Consensus sequence refers herein to the sequences AG/GT AGT found to occur at the exon-intron boundary (or donor sequence) and ( ) n N AG/G found to occur at the intron-exon boundary (or acceptor sequence). See Mount, S.M., Nucleic Acids Research 10(2) , 459-472 (1982). Analyses of the frequency with which individual bases occur in particular positions yielded a consensus sequence for the donor and acceptor sequences. It is also known that introns begin with GT and end with AG. Breathnach, R. et al ., PNAS (USA) 75 , 4853-4857 (1978).
  • Control region refers to specific sequences at the 5 ⁇ and 3 ⁇ ends of eukaryotic genes which may be involved in the control of either transcription or translation. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription is a CXCAAT region where X may be any nucleotide. At the 3 ⁇ end of most eukaryotic genes is an AATAAA sequence which may be the signal for addition of the polyadenylation tail to the 3 ⁇ end of the transcribed mRNA.
  • Promoter refers to the nucleotide segment recognized by RNA polymerase molecules that start RNA synthesis. Promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. beta actin promoter.
  • the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication. Fiers et al ., 1978, “Nature", 273 : 113.
  • the immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. Greenaway, P.J. et al ., Gene 18 , 355-360 (1982). Of course, promoters from the host cell or related species also are useful herein.
  • Enhancer refers to cis-acting elements of DNA, usually about from 10-300 bp, that act on a promoter to increase its transcription. Transcription of a DNA encoding a desired heterologous protein by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are relatively orientation and position independent having been found 5 ⁇ (Laimins, L. et al ., PNAS 78 , 993 [1981]) and 3 ⁇ (Lusky, M.L., et al ., Mol. Cell Bio. 3 , 1108 [1983]) to the transcription unit, within an intron (Banerji, J.L.
  • enhancer sequences are now known from mammalian genes (globin, elastase, albumin, ⁇ -fetoprotein and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding the desired heterologous protein. The 3 ⁇ untranslated regions also include transcription termination sites.
  • Selection genes may contain a selection gene, also termed a selectable marker.
  • a selection gene encodes a protein, sometimes referred to as a secondary protein, necessary for the survival or growth of a host cell transformed with the vector.
  • suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase or neomycin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure.
  • DHFR dihydrofolate reductase
  • thymidine kinase thymidine kinase
  • neomycin thymidine kinase
  • CHO DHFR ⁇ cells and mouse LTK ⁇ cells. These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media.
  • An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells which were not transformed with the DHFR or TK gene will not be capable of survival in non-supplemented media. Therefore, direct selection of those cells requires cell growth in the absence of supplemental nutrients.
  • the second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, Southern P. and Berg, P., J. Molec. Appl. Genet. 1 , 327 (1982), mycophenolic acid, Mulligan, R.C. and Berg, P. Science 209 , 1422 (1980) or hygromycin, Sugden, B. et al ., Mol. Cell. Biol. 5 :410-413(1985).
  • the three examples given above employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug neomycin (G418 or geneticin), xgpt (mycophenolic acid) or hygromycin, respectively.
  • G418 or geneticin neomycin
  • xgpt mycophenolic acid
  • hygromycin hygromycin
  • Amplification refers to the increase or replication of an isolated region within a cell's chromosomal DNA. Amplification is achieved using a selection agent e.g. methotrexate (MTX) which inactivates DHFR. Amplification or the making of successive copies of the DHFR gene results in greater amounts of DHFR being produced in the face of greater amounts of MTX. Amplification pressure is applied notwithstanding the presence of endogenous DHFR, by adding ever greater MTX to the media. Amplification of a desired gene can be achieved by cotransfecting a mammalian host cell with a plasmid having a DNA encoding a desired protein and the DHFR or amplification gene so that cointegration can occur.
  • MTX methotrexate
  • Preferred suitable host cells for expressing the vectors of the instant invention encoding the desired heterologous proteins in higher eukaryotes include: monkey kidney CVI line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293, Graham, F.L. et al . J. Gen Virol. 36 , 59 [1977]); baby hamster kidney cells (BHK, ATCC CCL 10); chinese hamster ovary-cells-DHFR (described by Urlaub and Chasin, PNAS (USA) 77 , 4216, [1980]); mouse sertoli cells (TM4, Mather, J.P., Biol. Reprod.
  • monkey kidney cells CVI ATCC CCL 70); african green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); rat hepatoma cells (HTC, M1.54, Baumann, H. et al ., J. Cell Biol. 85 , 1-8 [1980]); and, TRI cells (Mather, J.P. et al ., Annals N.Y. Acad. Sci. 383 , 44-68 [1982]).
  • Transformation means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integration. Unless otherwise provided, the method used herein for transformation of the host cells is the method of Graham, F. and van der Eb, A., Virology 52 , 456-457 (1973).
  • Host cells may be transformed with the expression vectors of the instant invention and cultured in conventional nutrient media modified as is appropriate for inducing promoters, selecting transformants or amplifying genes.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • Transfection refers to the taking up of an expression vector by a host cell whether or not any coding sequences are in fact expressed. Numerous method of transfection are known to the ordinarily skilled artisan, for example, CaPO4 and electroporation. Successful transfection is generally recognized when any indication of the operation of this vector occurs within the host cell. However, in the context of the present invention successful transfection refers to stable continuous expression of a desired heterologous protein by a host culture over numerous generations.
  • Choosing of the host production cell is achieved by screening for transient expression and then unamplifed expression using the method of the instant invention.
  • Vectors were screened for transient expression to determine which vectors could be used to express a desired heterologous protein.
  • Transient expression provides an indication of whether the particular plasmid that has been taken up functions, i.e., is transcribed and translated to produce the desired protein. During this time the plasmid DNA which has entered the cell is transferred to the nucleus. The DNA is in a nonintegrated state, free within the nucleus. Transcription of the plasmid taken up by the cell occurs during this period. Vectors which were identified as capable of producing the desired heterologous protein transiently were then used to establish a stable continuous production cell.
  • Transient expression refers to a short period (12-72 hrs) following transfection. Following this initial period after transfection the plasmid DNA becomes degraded or diluted by cell division. Random integration within the cell chromatin occurs. Screening the cells after two to three weeks of unamplified expression is an indicia of cells which have retained the recombinant DNA leading to a permanent cell line.
  • Coatest Factor VIII was purchased from Helena Laboratories, Beaumont, TX (Cat. No. 5293). The procedure used was essentially that provided by the manufacturer for the "end point method" for samples containing less than five percent protein.
  • Production cell lines were established using plasmids of the instant invention which were shown to function transiently in a wide variety of cells.
  • Expression vectors were transfected into a number of cell lines. For these transfections a total of 10 ⁇ g of DNA/ml precipitate were used. Selection for expression was made possible in these cells using a selectable marker as described above. All cells were transfected with modified CaPA4 technique except BRL cells which were found to be sensitive to calcium. Electroporation was used with these cells. Transfected cells were selected from suitable host cells as previously described.
  • the protocol used to establish production cell lines relied heavily on the staining method described above. Two days following transfection, cells were subcultured into a selection media. Media was titrated for the proper amount of the particular substance needed for selection. At the same time that cells were transfected to establish a production cell line, a dish of each cell type was assayed for transient expression.
  • Plasmids are designated by a lower case p preceded and/or followed by capital letters and/or numbers.
  • the starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures.
  • equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
  • “Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences, restriction sites, in the DNA.
  • the various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan.
  • typically 1 ⁇ g of plasmid or DNA fragment is used with about 2 units of enzyme in about 2 ⁇ l of buffer solution.
  • For the purpose of isolating DNA fragments for plasmid construction typically 5 to 10 ⁇ g of DNA would be digested with 20 to 40 units of enzyme in a larger volume.
  • Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about one hour at 37°C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction was run directly on a gel to isolate the desired fragment.
  • Dephosphorylation refers to the removal of the terminal 5 ⁇ phosphates by treatment with bacterial alkaline phosphatase (BAP). This procedure prevents the two restriction cleaved ends of a DNA fragment from "circularizing” or forming a closed loop that would impede insertion of another DNA fragment at the restriction site. Procedures and reagents for dephosphorylation are conventional. Maniatis, T. et al ., 1982, Molecular Cloning pp. 133-134. Reactions using BAP are carried out in 50mM Tris at 68°C to suppress the activity of any exonucleases which may be present in the enzyme preparations. Reactions were run for one hour. Following the reaction the DNA fragment is gel purified.
  • BAP bacterial alkaline phosphatase
  • Oligonucleotides refers to short length single or double stranded polydeoxynucleotides which are chemically synthesized by known methods and then purified on polyacrylamide gels.
  • Ligase refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T. et al ., Id ., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase ("ligase”) per 0.5 ⁇ g of approximately equimolar amounts of DNA fragments to be ligated.
  • ligase T4 DNA ligase
  • “Filling” or “blunting” refers to the procedures by which the single stranded end in the cohesive terminus of a restriction enzyme-cleaved nucleic acid is converted to a double strand. This eliminates the cohesive terminus and forms a blunt end. This process is a versatile tool for converting a restriction cut end that may be cohesive with the ends created by only one or a few other restriction enzymes into a terminus compatible with any blunt-cutting restriction endonuclease or other filled cohesive terminus.
  • blunting is accomplished by incubating 2-­15 ⁇ g of the target DNA in 10mM MgCl2, 1mM dithiothreitol, 50mM NaCl, 10mM Tris (pH 7.5) buffer at about 37°C in the presence of 8 units of the Klenow fragment of DNA polymerase I and 250 ⁇ M of each of the four deoxynucleoside triphosphates.
  • the incubation generally is terminated after 30 min. phenol and chloroform extraction and ethanol precipitation.
  • Northern blotting is a method by which the presence of a cellular mRNA is confirmed by hybridization to a known, labelled oligonucleotide or DNA fragment.
  • Northern analysis shall mean electrophoretic separation of the mRNA on 1 percent agarose in the presence of a denaturant (formaldehyde 7%), transfer to nitrocellulose hybridization to the labelled fragment as described by Maniatis, T. et al ., Id., p. 202.
  • the cDNA encoding human factor VIII was used in the construction of plasmids which would direct the expression of factor VIII protein in transfected mammalian cells (Wood, W. et al ., Nature [Lond.] 312 :330-337 [1984]). Those transformed mammalian cells secreted approximately .14 mU/ml of factor VIII. The instant method provides continuous production of factor VIII with yields significantly greater.
  • FIG 1 shows the steps for construction of the factor VIII expression vector used to establish production cell lines for factor VIII. The three parts of the construction are detailed below.
  • the ampicillin resistance marker and replication origin of the final vector was derived from the starting plasmid pUC13pML a variant of the plasmid pML (Lusky, M. and Botchen, M., Nature 293 , 79 [1981]).
  • pUC13pML was constructed by transferring the polylinker of pUC13 (Veira, J. and Messing, J., Gene 19 :259(1982)) to the Eco RI and Hin dIII sites of pML.
  • a second starting plasmid pUC8CMV was the source of the CMV enhancer, promoter and splice donor sequence.
  • pUC8CMV was constructed by inserting nucleotides 1 through 732, shown in Figure 17, for the CMV enhancer, promoter and splice donor sequence into the blunted Pst I and Sph I sites of pUC8. Veira, J. and Messing, J. supra .
  • Synthetic Bam HI- Hin dIII linkers (commercially available from New England Biolabs) were ligated to the cohesive Bam HI end creating a Hin dIII site. Following this ligation a Hin dIII- Hin cII digest was performed. This digest yielded a fragment of approximately 800bp which contained the CMV enhancer, promoter and splice donor site.
  • This 800bp fragment was ligated to a 2900bp piece of pUC13pML.
  • the fragment required for the construction of pF8CIS was obtained by digestion of the above intermediate plasmid with Sal I and Hin dIII.
  • This 3123bp piece contained the resistance marker for ampicillin, the origin of replication from pUC13pML and the control sequences for the CMV including the enhancer, promoter and splice donor site.
  • the Ig variable region intron and splice acceptor sequence was constructed using a synthetic oligomer as shown in the central portion of Figure 1.
  • a 99 mer and a 30 mer were chemically synthesized having the following sequence for the IgG intron and splice acceptor site (Bothwell et al ., 1981):
  • DNA polymerase I (Klenow fragment) filled in the synthetic piece and created a double stranded fragment. Wartell, R.M. and W.S. Reznikoff, Gene 9 , 307 (1980). This was followed by a double digest of Pst I and Hin dIII. This synthetic linker was cloned into pUC13 (Veira, J. and Messing, J., Gene 19 , 259 [1982]) at the Pst I and Hin dIII sites. The clone containing the synthetic oligonucleotide, labelled pUCIg.10, was digested with PstI. A Cla I site was added to this fragment by use of a Pst I- Cla I linker. Following digestion with Hin dIII a 118bp piece containing part of the Ig intron and the Ig variable region splice acceptor was gel isolated.
  • the third part of the construction scheme replaced the hepatitis surface antigen 3 ⁇ end with the polyadenylation site and transcription termination site of the early region of SV40.
  • a vector, pUC.SV40 containing the SV40 sequences was inserted into pUC8 at the Bam HI site described in Viera, J. and Messing, J., supra .
  • pUC.SV40 was then digested with Eco RI and Hpa I.
  • a 143bp fragment containing only the SV40 polyadenylation site was gel isolated from this digest. Two additional fragments were gel isolated following digestion of pSVE.8c1D. European Patent Publication No. 150,457.
  • the 4.8 kb fragment generated by Eco RI and Cla I digest contains the SV40-DHFR transcription unit, the origin of replication of pML and the ampicillin resistance marker.
  • the 7.5 kb fragment produced following digestion with Cla I and Hpa I contains the cDNA for factor VIII.
  • a three-part ligation yields pSVE.8c24D.
  • This intermediate plasmid was digested by Cla I and Sal I to give a 9611bp fragment containing the cDNA for factor VIII with an SV40 polyadenylation and transcription termination sites followed by the SV40 DHFR transcription unit.
  • the final three part ligation to yield pF8CIS used: a) the 3123bp Sal I Hin dIII fragment containing origin of replication, the ampicillin resistance marker and the CMV enhancer, promoter and splice donor; b) The 118bp Hin dIII- Cla I fragment containing the Ig intron and splice acceptor; and, c) a 9611bp Cla I- Sal I fragment containing the cDNA for factor VIII, SV40 polyadenylation site and the SV40 DHFR transcription unit.
  • a portion of the sequence of the expression vector pF8CIS is shown in Figure 17.
  • the vector pF8CSSS containing the cytomegalovirus enhancer and promoter, an engineered stabilizing sequence, the cDNA encoding factor VIII and the SV40 polyadenylation site was constructed.
  • the entire intron region including donor and acceptor sequences was deleted and replaced by an engineered stabilizing sequence.
  • the stabilizing sequence is a synthetic double stranded oligomer having a sequence of the mature mRNA following splicing.
  • the stabilizing sequence was inserted between the unique Sac II- Cla I sites of pF8CIS.
  • the sequences of the synthetic oligomers are as follows:
  • the synthetic oligomers comprise the appropriate nucleotides of the donor and acceptor consensus splice sequences.
  • the juxtaposition of the splice donor sequence to the splice acceptor sequence is indicated by the underline.
  • This vector resembles the pF8CIS vector discussed above except for the deletion of the intron portion and replacement with an engineered stabil­izing sequence. This construction eliminates the actual splicing of the noncoding region from recently the transcribed mRNA.
  • a portion of the sequence of the expression vector pF8CSSS containing the engineered stabilizing sequence is shown in Figure 19.
  • the vector pF8SCIS containing the SV40 enhancer and promoter, the cytomegalovirus splice donor site and a portion of the intron, the Ig intron and splice acceptor site, the cDNA encoding factor VIII and the SV40 polyadenylation and transcription termination sites were constructed.
  • Figure 2 shows the construction of pF8SCIS.
  • This vector was constructed using a three part ligation. The preparation of each of the three fragment of DNA used in this ligation is described below:
  • the first fragment contained the SV40 early region promoter and enhancer and one half the ampicillin resistance marker which was obtained from plasmid pML.
  • the starting plasmid for the first of three fragments was pAML3P.8Cl. European Patent Publication No. 160,457.
  • This plasmid was cut with Sac I. Using the whole enzyme DNA polymerase I this 3 ⁇ overhang created by Sac I was blunted. Following this reaction the plasmid was cut with Pvu I. The desired 434bp fragment was isolated from an acrylamide gel.
  • the second and third fragments used in this construction were isolated from the plasmid pF8CIS which is described above.
  • Fragment 2 contained the splice donor from CMV immediate early gene and part of the following intron and the intron and splice acceptor synthetically made as described above.
  • pF8CIS was cut with SacII and the resulting 3 ⁇ overhang was blunted by the use of DNA polymerase I. This reaction was followed by cleavage with Cla I. Since the sequence surrounding the Cla I site in pF8CIS prevents cleavage if the plasmid is grown in a methylation plus strain, pF8CIS was prepared from dam ⁇ strain GM48. Marinus, M.G. and Maris, N.R., Bacteriol. 114 , 1143-1150 (1973) and Geier, G.E. and Madrid, P., J. Biol. Chem. 254 , 1408-1413 (1979). Since both Sac II and Cla I are unique sites in this vector the 231bp fragment was easily isolated from an agarose gel.
  • the third fragment contains the cDNA for factor VIII, SV40 early region polyadenylation site, a SV40-DHFR transcriptional unit, the origin of replication of pML and half of the ampicillin gene.
  • the 11308bp fragment was prepared by digestion of pF8CIS (dam ⁇ ) with Cla I and Pvu I.
  • the three part ligation creating pF8SCIS destroys the SacI and SacII sites, maintains the Cla I site and reconstructs the amp r gene at the Pvu I site.
  • a portion of the nucleotide sequence of the expression vector pF8SCIS is shown in Figure 18.
  • Factor VIII expression was assayed based on immunoperoxidase staining of transfected cells. Gorman et al. Cell 42 , 519-526 (1985). This assay was used to test vectors for the expression of factor VIII. Twelve monoclonal antibodies specific for factor VIII were screened for use in this assay. BHK 31A3B cells (European Patent Publication No. 160,457) were stained and compared with parental BHK line to screen cells which produce factor VIII. Monoclonal antibody BH6 was found to give the strongest signal with the least amount of background. Transfections were performed and transient expression of factor VIII was assessed. Cells (Cos, 293, CHO, BHK, TM4) were transfected using the CaPO4 technique.
  • a second antibody of rabbit anti-mouse IgG was diluted in PBS + fetal calf serum at a dilution of 1:150. A two hour incubation was followed by another series of washes.
  • Ortho-diansidine (Sigma) was used as a substrate for developing the peroxidase reagent. A ethanol saturated solution of ortho-diansidine was diluted 1:100 in PBS with 1:10,000 dilution of hydrogen peroxide. This substrate was left on the cells for 2 hrs at room temperature or overnight at 4°C.
  • This method provided a screen for those factor VIII vectors directing factor VIII expression. This method determines transient factor VIII expression. Staining thirty-six hours after transfection provides an indication of whether the vector was transcribed and the mRNA translated.
  • pF8CIS directed transient expression of factor VIII in at least five different cell lines: COS, 293, CHO, TM4 and BHK.
  • Figure 3A shows transient expression of the vector pF8CIS in CHO cells.
  • FIG. 3B shows transient expression of the vector pF8SCIS in CHO cells. Since the CMV enhancer and promoter can be completely replaced by the analogous SV40 enhancer and promoter, factor VIII production is not dependent on the specific transcriptional start signal but rather is dependent on other parts of the control region such as the stabilizing sequence site in the vector.
  • results of the transient expression screen for factor VIII produced two classes of cells: those cell types which stained positively for factor VIII thirty-six hours after transfection (Category 1); and, those cell types having no detectable transient expression of factor VIII (Category 2).
  • the host cells comprising each category are indicated below:
  • stabilizing sequence is important. For example location of an intron 3 ⁇ to the cDNA encoding factor VIII failed to express factor VIII.
  • the splice donor-acceptor arrangement containing the CMV splice donor sequence and a chimeric intron comprising CMV sequences and the synthesized Ig variable region intron and acceptor is an example of a stabilizing sequence which will lead to the establishment of a cell line providing continuous production of factor VIII.
  • Production cell lines were established by transfecting the plasmids, containing a stabilizing sequence and shown to function transiently in a wide variety of cells, into a number of cell lines. For these transfections a total of 10 ⁇ g of DNA/ml precipitate was used. For transfection of CHO DHFR cells 4 ⁇ g of factor VIII plasmid was added to 6 ⁇ g salmon sperm DNA, which served as a carrier. Wigler, M. et al ., supra . Direct selection for expression of DHFR gene was possible in these cells. All other cell types required cotransfection with a plasmid expressing neomycin gene. Davies, J. and Jenning, A., Am. J. Trop. Med. Hyg.
  • the protocol used to establish production lines relied heavily on the staining method described above. Two days following transfection cells were subcultured into selection media lacking glycine, hypoxanthine and thymidine for the CHO DHFR cells or G418 containing media. Levels of G418 were titrated for the proper amount needed for selection.
  • factor VIII was detectable in CHO cells. Even at this high level of amplification, activity of factor VIII was low, 63 mU/ml. Continued amplification did not lead to increased production in CHO cell lines. Morphological analysis of three separately derived CHO lines show the cells staining for factor VIII to be enlarged in size and flattened. Fig. 5. Transformed CHO cells amplified to 10 ⁇ M produced no more than 200 mU/ml. These results indicate that the choice of a host cell is an important step in the establishment of a production cell system for factor VIII. Presently TM4, HTC and 293 have been used to establish permanent cell lines providing continuous production of factor VIII, thus qualifying as production cell lines.
  • TM4 cells Serum free media that had been conditioned for 48 hours by TM4 cells transfected with pF8CIS was assayed for factor VIII. As shown in Table 1 TM4 culture media shortened the clotting time of hemophilic plasma. Most of this coagulant activity was neutralized when TM4 media was preincubated with a polyclonal antibody against human factor VIII. Table 1. Secretion of active factor VIII from TM4 cells. TM4 media was either preincubated with 10 ⁇ g of a human factor VIII polyclonal antibody or with no addition for 30 minutes at 37°C. Subsequently the media was diluted 1:1 with 0.05 M Tris pH 7.5 containing 0.01% BSA and assayed by coagulation analysis. Purified plasma derived factor VIII (pd VIII) was treated similarly.
  • variants can be produced in cell culture to allow for specific modification in protein function.
  • Three variants were engineered.
  • the native factor VIII single chain 300,000 dalton protein is cleaved to subunits of 90,000 and 80,000 dalton which in turn are cleaved to the active subunits of 50,000, 43,000 and 73,000 dalton.
  • the B domain between amino acid 742 through 1648 has no defined function.
  • the same cell systems described for expression of the full length recombinant factor VIII protein were used to express the mutant.
  • the eukaryotic expression vector used to express the factor VIII fusion protein included: the enhancer (Boshart et al ., supra ), and promoter (Thomsen et al ., supra ) of the human cytomegalovirus (CMV) immediate early gene; the splice donor sequence located 3 ⁇ of the transcription initiation site of this gene (Boshart et al ., supra , Stenberg et al ., supra ); and a synthetic splice acceptor site from the mouse immunoglobulin variable region (Bothwell et al ., supra ).
  • CMV cytomegalovirus
  • the new coding region is flanked on the 3 ⁇ end by the SV40 early polyadenylation sequence and transcription termination site (Fiers et al ., supra ).
  • the vector includes an amplifiable marker, the SV40-DHFR transcription unit.
  • a 6761bp promoter containing fragment of pAML3P.D22 (European Patent Publication No. 160,457).
  • the fusion in the factor VIII gene was made by ligating the filled in Tth 111 I and Bam HI (amino acid 1563) sites within the factor VIII gene.
  • Figure 6 shows ligation of a 2516bp fragment of pAML3P.8cl (European Patent Publication No. 160,457) and a 11991bp fragment of pAML3P.8c9 to construct pAML3P.8L19 containing the fused region. This fusion was confirmed by DNA sequence.
  • a 4722bp Cla I- Xba I fragment containing the fusion region was cloned into a 5828bp Cla I- Xba I fragment of pF8CIS containing the CMV promoter-enhancer expression vector.
  • the CMV fragment was obtained from a dam ⁇ strain of E . coli where methylation does not prevent cutting at the Cla I site.
  • Example 2 The method described in Example 2 was applied to expression of the factor VIII variant which deleted nucleotides 796 through 1562, pF8CIS9080.
  • the 90kd + 142aa + 80kd fusion protein is expressed at higher levels than the full length protein.
  • the 90kd + 142aa + 80kd fusion protein is expressed at higher levels than the full length protein.
  • TM4 cells transfected with pF8CIS9080 showed both transient and stable expression of the fusion protein.
  • TM4 cells transfected with pF8CIS9080 showed a five-fold increase in the levels of the fusion protein as compared to the full length factor VIII.
  • HTC cells showed a similar enhancement in expression of the fusion factor VIII as compared to the full length factor VIII.
  • Fusion protein factor VIII is quite high in 293 cells as compared to full length factor VIII expression.
  • the unamplified population levels of 85 mU/104 cells/day were routinely achieved.
  • Expression levels of full length factor VIII were lower than the fusion factor VIII yielding 2.5 mU/104 cells/day. Since the control signals are identical in the pF8CIS and pF8CIS9080, the difference in expression levels must lie within the capability of the cell to produce full length message and/or protein.
  • the fusion protein was detected at an earlier point of amplification (100nM) than the full length (1000 nM), however as shown in figure 7 these cells were burdened by the expression of the fusion protein.
  • CHO cells expressing 90kd + 142aa + 80kd at 100 nM produced 0.5 ⁇ U/104 cells/day. Continued amplification was difficult due to mixed population seen in figure 8.
  • Clones selected at 1 ⁇ M MTX and 5 ⁇ M MTX showed no higher expression levels than the 0.1 ⁇ M MTX lines.
  • certain host cells were particularly adept at expression of factor VIII or its variants e.g. TM4.
  • Other host cells were of an intermediate nature in that the variant is expressed while the full length factor VIII is expressed in low levels or not at all e.g. 293 cells. A final group of host cells is unlikely to produce sufficient factor VIII for production, e.g. TRI.
  • the 90kd + 142aa + 80kd fusion was purified from 293 media using techniques previously described for full length recombinant factor VIII, Eaton, D.E. et al ., Biochemistry 25 , 505-512 (1986).
  • the purified fusion had a specific activity of 4,000-6,000 units/mg, which is comparable to the specific activity of plasma derived factor VIII.
  • SDS-PAGE the fusion resolved into two major bands with M r of 115,000 and 80,000.
  • a band with a M r of 180,000 was also seen and probably represents the single chain form of the fusion.
  • the M r 180,000, 115,000, and 80,000 proteins were all detected by a factor VIII polyclonal antibody in a Western blot ( Figure 11).
  • Coagulant activity of the 90kd + 142aa + 80kd fusion was activated 10-20 fold by thrombin ( Figure 12). This activation correlated with the generation of subunits with M r 50,000, 43,000, and 73,000 (Id.). Since factor VIII circulates in plasma bound to von Willebrands factor (vWF), binding of the 90kd + 142aa + 80kd fusion to vWF was also tested. Purified 90kd + 142aa + 80kd fusion that was passed over a vWF-Sepharose column quantitatively bound to the column ( Figure 13). Subsequently the fusion was eluted from the column with 0.25 M CaCl2, which is known to dissociate factor VIII-vWF complexes.
  • vWF von Willebrands factor
  • the above data show that the 90kd + 142aa + 80kd fusion expressed and secreted from the 293 cells was functionally similar to plasma derived factor VIII.
  • the 90kd + 142aa + 80kd fusion shortened the clotting time of hemophilic plasma and its activity was neutralized by a factor VIII antibody.
  • the fusion was activated and processed by thrombin similarly to plasma derived factor VIII.
  • the 90kd + 142aa + 80kd fusion also bound to vWF immobilized on Sepharaose and was dissociated from vWF under conditions known to dissociate factor VIII-vWF complexes.
  • Serum free media that had been conditioned for 48 hrs. by 293 cells transfected with pF8CIS9080 was assayed for factor VIII coagulant activity by coagulation analysis. After the media was diluted 1/50 it was assayed and found to shorten the clotting time of hemophilic plasma from 120 sec. to 58.9 sec., corresponding to 5.5 units/ml of factor VIII coagulant activity. This activity was neutralized by a polyclonal antibody against plasma derived factor VIII (Table 2).
  • Hemophilic plasma 50 ⁇ l was incubated with 50 ⁇ l of platelin (General Diagnostics) for 8 min. at 37°C. Subsequently 50 ⁇ l of media that had been diluted 1/50 with 0.05 M Tris pH 7.4 buffer containing 0.01 percent BSA was added and incubated 30 sec. CaCl2 (25 mM), 50 ⁇ l, was added and the clot time was measured. Media obtained from the parent cell line, which was not transfected, was not diluted. Antibody neutralization experiments were performed by preincubating undiluted media (100 ⁇ l) with 10 ⁇ g of factor VIII polyclonal antibody for 30 min. at room temp. The media was then diluted and assayed.
  • Activated protein C a plasma protein
  • APC Activated protein C
  • the arginine at position 336 can be changed to another amino acid, for example, lysine or glutamic acid.
  • Two vectors, pF8CIS336E and pF8CIS9080-336E, were constructed to determine whether position 336 was a site of inactivation.
  • pF8CIS336E and pF8CIS9080-336E were constructed to determine whether position 336 was a site of inactivation.
  • in vitro mutagenesis Naorris, K. et al ., Nucleic Acids Research, 11 , 5103-5112 [1983]
  • the arginine at position 336 was mutated to a glutamic acid (Fig.
  • Construction of pF89080-336E proceeded via another three part ligation as shown in Figure 10.
  • a 1115 bp SpeI-BglII fragment containing the 336E variant amino acid was transferred to create another variant fusion protein by ligation to a 891 bp Sac II- Spe I fragment and a 8564bp Bg1 II- Sac II fragment isolated from pF8CIS9080.
  • the Vector pCIHRX contained the cytomegalovirus enhancer and promoter, the cytomegalovirus splice donor site, the Ig variable region splice acceptor site, the cDNA encoding H2 prepro­relaxin and the hepatitis surface antigen polyadenylation and transcription termination sites.
  • Figure 14 shows the steps for construction of the prorelaxin vector. The same intron and splice acceptor sequence described previously from the Ig variable region was maintained. 677bp of the preprorelaxin cDNA followed these 5 ⁇ processing signals. While the 5 ⁇ control signals were identical to pF8CIS the polyadenylation region and termination sequence signals were from the hepatitis surface antigen gene rather than SV40.
  • the plasmid pSVERX (see copending U.S. patent application U.S.S.N. 06/907,197, filed September 12, 1986, and corresponding European application) was cut with Hin dIII to isolate a 1700bp fragment containing the pre-prorelaxin cDNA followed by the hepatitis B surface antigen (HBsAg) 3 ⁇ polyadenylation site.
  • HBsAg hepatitis B surface antigen
  • a Kpn I site was 3 ⁇ to the HBsAg polyadenylation site and 5 ⁇ to the start of the SV40 early promoter which in this vector was used to drive expression of the DHFR cDNA.
  • This Hin dIII fragment was inserted into pML linearized at the Hin dIII site. Reclosures were minimized by treatment with bacterial alkaline phosphatase (BAP). Ampicillin resistant colonies were screened to isolate clones which had inserted the pre-prorelaxin gene so that the 5 ⁇ end of the gene was next to the Cla I site of pML.
  • BAP bacterial alkaline phosphatase
  • the intermediate plasmid pClARX was cut with Cla I and Kpn I to isolate a 1360bp fragment containing the pre-prorelaxin gene followed by the hepatitis surface antigen 3 ⁇ polyadenylation sequences. This fragment was ligated to the 5143bp fragment created by cutting pF8CIS dam ⁇ with Cla I and Kpn I.
  • pCISRX The two starting vectors for this construction are pCIHRX and pF8CIS.
  • Sac II was used to cleave 3 ⁇ of the cDNA. The resultant 3 ⁇ overhang was blunted by T4 polymerase.
  • pCIHRX was then cut with Bam HI.
  • This site separates the chimeric intron from the 5 ⁇ end of the relaxin gene.
  • An 861bp fragment was gel isolated from the BamHI treatment.
  • the SV40 polyadenylation site, DHFR, transcription unit, bacterial origin of replication and amp r gene, as well as the CMV enhancer and promoter and splice donor were isolated from pF8CIS. These elements were isolated in two fragments, as a 2525bp Sal I- Bam HI fragment and a Hpa I- Sal I 3113 bp fragment.
  • a three part ligation of the Bam HI- Sac II (blunted) fragment with the Hpa I- Sal I fragment and Sal I to Bam HI fragment yields pCISRX.
  • the expression capabilities of the two relaxin expression vectors pCIHRX and pCISRX were assayed using several anti-relaxin antibodies in the immunoperoxidase method described above. Three rabbit polyclonals and three mouse monoclonal antibodies were tested on COS cells transfected with pSVERX. One monoclonal RX-I was found to give intense staining with no background.
  • pCIHRX and pCISRX were tested for prorelaxin expression and compared to pSVERX.
  • pCIHRX and pCISRX vectors differed in the polyadenylation sequence.
  • pCIHRX contained the hepatitis surface antigen polyadenylation sequence while pCISRX contained the SV40 early region polyadenylation sequence.
  • TM4 and CHO cells were transfected with 10 ⁇ g total DNA which included 1 ⁇ g pRSVneo, 5 ⁇ g salmon sperm carrier and 4 ⁇ g of plasmids pSVERX, pCIHRX and pCISRX. Cells were glycerol shocked as described above. Thirty-six hours following transfection cells were fixed and stained with IH6 to identify transformed cells making prorelaxin. Positive staining cells were seen in 293 and TM4 cells transfected with pCIHRX and pCISRX. Duplicate plates of CHO, 293 and TM4 cells were split and subjected to the staining protocol described above to screen for prorelaxin production cells.
  • the vector pCIHt-PA containing the cytomegalovirus enhancer and promoter, the cytomegalovirus splice donor site and intron, the Ig variable region splice acceptor site, the cDNA encoding t-PA (Pennica et al ., Nature 301 , 214 (1983)) and the hepatitis surface antigen polyadenylation and transcription termination site was constructed.
  • Figure 16 shows the steps for construction of the t-PA vector.
  • the t-PA cDNA was first cloned into pML to provide a Cla I site at the 5 ⁇ end of the gene. To do this a 3238 bp Hin dIII fragment from pSVpa-DHFR (otherwise referred to as pETPFR in UK patent 2,119,804 B) was inserted into the Hin dIII site of pML. Colonies were screened for clones which have the 5 ⁇ end of the cDNA juxtaposed to the Cla I site. The intermediate plasmid labelled pCLAt-PA is shown in Figure 16. A t-PA cDNA followed by the 3 ⁇ polyadenylation region was isolated as a Cla I- Kpn I fragment of 2870bp.
  • This fragment was ligated to the 5146bp fragment of pF8CIS.
  • This Cla I- Kpn I fragment of the CIS vector provided the 5 ⁇ control region, a SV40-DHFR transcriptional unit, the ampicillin resistance gene and origin region from pML.
  • pCIHt-PA is analogous to pCIHRX, discussed above, with the exception of the cDNA coding for the desired heterologous gene.
  • t-PA Expression levels of t-PA were compared by transfecting CHO and 293 cells with pSVpaDHFR, pCMVt-PA and pCIHt-PA.
  • the former two vectors did not contain a stabilizing sequence and thus served as controls for the vector pCIHt-PA containing the cDNA encoding t-PA constructed in accord with the instant invention.
  • Media from each of the cultured transformed 293 cells were assayed and the following results were obtained: pSVpaDHFR gave 30 ng/ml; pCMVt-PA gave 200 ng/ml of t-PA; and pCIHt-PA gave 420 ng/ml of t-­PA.
  • the vector pCISt-PA containing the cytomegalovirus enhancer and promoter, the cytomegalovirus splice donor site and intron, the Ig variable region splice acceptor site, the cDNA encoding t-PA and the pSV40 polyadenylation sequence was constructed.
  • the starting vectors for this construction are pCIHt-PA and pF8CIS (see Figure 20).
  • the latter vector has the same 5 ⁇ controls as pCIHt-PA but includes the cDNA for factor VIII and the SV40 polyadenylation site. Sac II was used to cleave 3 ⁇ of the t-PA cDNA. The resultant 3 ⁇ overhang was blunted by T4 polymerase.
  • pCIHt-PA was then cut with Cla I. This site separates the chimeric intron from the 5 ⁇ end of the t-PA gene. A 2870bp fragment was gel isolated from the Cla I treatment.
  • the SV40 polyadenylation site, DHFR, transcription control, bacterial origin of replication and amp r gene, as well as the CMV enhancer and promoter and splice donor were isolated from pF8CIS. These elements were isolated into fragments as a 2525bp Sal I- Cla I fragment and a Hpa I- Sal I 3113 fragment. A three part ligation of the Sac II(blunt)- Cla I fragment with the Hpa I- Sal I fragment and Sal I- Cla I fragment yields pCISt-­PA.

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WO1991007497A1 (fr) * 1989-11-17 1991-05-30 Kabigen Ab Expression de proteines heterologues de cellules eucaryotes
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WO1992011380A1 (fr) * 1990-12-21 1992-07-09 Imperial Chemical Industries Plc Systemes d'expression
EP0364096A3 (fr) * 1988-09-06 1992-08-05 International Genetic Engineering, Inc. (Ingene) Eléments d'expression de gènes et production d'anticorps souris-humains chimériques
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WO1996004391A1 (fr) * 1994-08-05 1996-02-15 Genentech, Inc. Procede de selection de cellules hotes a haut niveau d'expression
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US5576184A (en) * 1988-09-06 1996-11-19 Xoma Corporation Production of chimeric mouse-human antibodies with specificity to human tumor antigens
US5688688A (en) * 1984-10-31 1997-11-18 Chiron Corporation Vector for expression of a polypeptide in a mammalian cell
EP0867514A3 (fr) * 1997-02-28 1999-12-08 Bayer Corporation Procédé pour augmenter la production des protéines
US6013857A (en) * 1989-12-01 2000-01-11 Pharming B.V. Transgenic bovines and milk from transgenic bovines
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GB2197321A (en) 1988-05-18
DE3730599A1 (de) 1988-07-07
IE872455L (en) 1988-03-12
EP0260148A3 (fr) 1989-06-07
JPS63152986A (ja) 1988-06-25
JP3148121B2 (ja) 2001-03-19
DK473987D0 (da) 1987-09-11
FR2603899A1 (fr) 1988-03-18
JPH09103296A (ja) 1997-04-22
NZ221790A (en) 1990-07-26
AU613316B2 (en) 1991-08-01
JP2888518B2 (ja) 1999-05-10
GB8721424D0 (en) 1987-10-21
FR2603899B1 (fr) 1990-07-13
DK175363B1 (da) 2004-09-13
GB2197321B (en) 1990-10-03
AU7831787A (en) 1988-05-19
DK473987A (da) 1988-03-13
JP2000308497A (ja) 2000-11-07

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